The filoviruses, Ebola and Marburg viruses (EBOV and MARV), are emerging, negative-strand RNA viruses associated with outbreaks of severe viral hemorrhagic fever. The virulence and emerging nature of these zoonotic pathogens makes them a significant threat to human health, potential agents of bioterrorism, and NIAID category A priority pathogens. Currently, no approved anti-filovirus therapeutics are available. Among the proteins encoded by EBOV, VP35 is of particular interest for antiviral development because it makes a critical contribution to pathogenesis through its role in viral replication and host immune suppression. The Basler lab first identified the VP35 protein as an EBOV-encoded inhibitor of the RIG-I signaling pathway, which normally functions to trigger IFN?/? production in response to RNAs produced during viral infection. VP35 also functions as a co-factor in the filoviral replication complex. Important insight into the molecular basis by which VP35 IFN inhibitory domain (IID) participates in the viral RNA polymerase complex and inhibits the RIG-I pathway have recently been provided by structural studies from the Amarasinghe laboratory. Our recent collaborative studies have identified a novel interaction between Ebola VP35 and NP proteins that will be exploited for high-throughput screen (HTS) development. Since we are targeting a protein-protein interface, we will use fragment screens to validate the interface. Studies here are aimed to provide highly sensitive assays that can be converted to HTS assays in order to identify replication inhibitors.